AMPK is well established as a sensor and regulator of cellular energy homeostasis (Hardie D. G. and Hawley S. A; “AMP-activated protein kinase: the energy charge hypothesis revisited” Bioassays, 23, 1112, (2001), Kemp B. E. et al. “AMP-activated protein kinase, super metabolic regulator”, Biochem; Soc. Transactions, 31, 162 (2003)). Allosteric activation of this kinase due to rising AMP levels occurs in states of cellular energy depletion. The resulting serine/threonine phosphorylation of target enzymes leads to an adaptation of cellular metabolism to low energy state. The net effect of AMPK activation induced changes is inhibition of ATP consuming processes and activation of ATP generating pathways, and therefore regeneration of ATP stores. Examples of AMPK substrates include acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (Carling D. et al. “A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis”, FEBS letters, 223, 217 (1987)). Phosphorylation and therefore inhibition of ACC leads to simultaneous decrease in fatty acid synthesis (ATP-consuming) and increase in fatty acid oxidation (ATP-generating). Phosphorylation and resulting inhibition of HMG-CoA reductase leads to a decrease in cholesterol synthesis. Other substrates of AMPK include hormone sensitive lipase (Garton A. J. et al. “Phosphorylation of bovine hormone-sensitive lipase by AMP-activated protein kinase; A possible antilipolytic mechanism”, Eur. J. Biochem. 179, 249, (1989)), glycerol-3-phosphate acyltransferase (Muoio D. M. et al. “AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target”, Biochem. J., 338, 783, (1999)), malonyl-CoA decarboxylase (Sarah A. K. et al. “Activation of malonyl-CoA decarboxylase in rat skeletal muscle by contraction and the AMP-activated protein kinase activator 5-aminoimidazole-4-caboxamide-1-beta-D-ribofuranoside”, J. Biol. Chem. 275, 24279, (2000)).
AMPK is also involved in the regulation of liver metabolism. Elevated glucose production by the liver is a major cause of fasting hyperglycemia in type 2 diabetes (T2D) (Saltiel et al. “New perspectives into the molecular pathogenesis and treatment of type 2 diabetes”, Cell 10, 517-529 (2001)). Gluconeogenesis in the liver is regulated by multiple enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase -G6Pase. Activation of AMPK suppresses the transcription of theses genes in hepatoma cells (Lochhead et al. “5-aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase”, Diabetes, 49, 896-903 (2000)).
AMPK activation also down-regulates gluconeogenesis acting on some other genes expression. These effects may be due to its ability to down-regulate key transcription factors such as SREBP-1c (Zhou G. et al., “Role of AMP-activated protein kinase in mechanism of metformin action” J. Clin. Invest., 108, 1167 (2001)), ChREBP (Kawaguchi T. et al., “Mechanism for fatty acids sparing effect on glucose induced transcription: regulation of carbohydrate response element binding protein by AMP-activated protein kinase” J. Biol. Chem. 277, 3829 (2001)), or HNF-4-alpha (Leclerc I. et al., “Hepatocyte nuclear factor-4alpha involved in type 1 maturity-onset diabetes of the young is a novel target of AMP-activated protein kinase” Diabetes, 50, 1515 (2001)) or to direct phosphorylate transcriptional coactivators such as p300 (Yang W et al., “Regulation of transcription by AMP-activated protein kinase; Phosphorylation of p300 blocks its interaction with nuclear receptors” J. Biol. Chem. 276, 38341 (2001)) or TORC2.
AMPK is considered as an attractive candidate for contraction-induced skeletal muscle glucose uptake because it is activated in parallel with elevation in AMP and a reduction in creatine phosphate energy stores (Hutber et al. “Electrical stimulation inactivates muscle acetyl-CoA carboxylase and increases AMP-activated protein kinase” Am. J. Physiol. Endocrinol. Metab. 272, E262-E66 (1997)). Furthermore, AICAR-induced activation of AMPK increases glucose uptake (Merrill et al. “AICA Riboside increases AMP-activated protein kinase, fatty acid oxidation and glucose uptake in rat muscle” Am. J. Physiol. Endocrinol. Metab. 273, E1107-E1112 (1997)) concomitantly with glucose transporter 4 (GLUT4) fusion with plasma membrane (Kurth-Kraczek “5′-AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle”, Diabetes, 48, 1667-1671 (1999)). Over-expression of an alpha2 kinase dead subunit in skeletal muscle abolishes AICAR, but partially impairs contraction-stimulated glucose uptake (Mu J. et al. “A role for AMP-activated protein kinase in contraction and hypoxia-regulated glucose transport in skeletal muscle”, Mol. Cell. 7, 1085-1094 (2001)). These findings suggest that additional pathways mediate contraction induced glucose uptake, whereas it is clear that AMPK mediates the effects of AICAR on glucose uptake.
Despite extensive studies on upstream stimuli that activate AMPK, investigation on the downstream substrate(s) of AMPK-mediated glucose uptake is lacking. More recent reports revealed that Akt substrate of 160 kDa (AS160) is an important substrate downstream of Akt that is involved in insulin-stimulated glucose uptake. In addition to insulin, contraction and activation of AMPK by AICAR is associated with increased phosphorylation of AS160 in rodent skeletal muscle. Phosphorylation of AS160 is impaired or abolished in skeletal muscle from AMPK a2 knockout, g3 knockout, and a2-kinase dead mice in response to AICAR treatment (Treeback et al. “AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits”, Diabetes (2006)). This corroborates findings of impaired AICAR-stimulated glucose uptake in skeletal muscle of such mice (Jorgensen S. B. et al. “Knockout of the a2 but not a1 5′-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1b-4 ribofuranoside but not contraction-induced glucose uptake in skeletal muscle”, J. Biol. Chem. 279, 1070-1079 (2004)). Therefore, AS160 appears to be a downstream target of AMPK in mediating glucose uptake in skeletal muscle.
Taken together, all these metabolic effects evidence that AMPK suppresses liver gluconeogenesis and lipid production, while decreasing hepatic lipid deposition via increased lipid oxidation, thus improving the glucose and lipid profiles in T2D. More recently, involvement of AMPK in the regulation of not only cellular but also whole body energy metabolism has become apparent. It was shown that the adipocyte-derived hormone leptin leads to a stimulation of AMPK and therefore to an increase in fatty acid oxidation in skeletal muscle (Minokoshi Y. et al. “Leptin stimulates fatty-acid oxidation by activating AMP activated protein kinase”, Nature, 415, 339 (2002)). Adiponectin, another adipocyte derived hormone leading to improved carbohydrate and lipid metabolism, has been shown to stimulate AMPK liver and skeletal muscles (Yamanauchi T. et al. “Adiponectin stimulates glucose utilization and fatty acid oxidation by activating AMP-activated protein kinase”, Nature Medicine, 8, 1288, (2002), Tomas E. et al. “Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: Acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation” PNAS, 99, 16309, (2002)). The activation of AMPK in these circumstances seems independent of increasing cellular AMP levels but rather due to phosphorylation by one or more upstream kinases yet to be identified.
Based on the knowledge of the above-mentioned consequences of AMPK activation, deep beneficial effects would be expected from in vivo activation of AMPK. In liver, decreased expression of gluconeogenic enzymes would be expected to reduce hepatic glucose output and improve overall glucose homeostasis; both direct inhibition and/or reduced expression of key enzymes in lipid metabolism would be expected to increase glucose uptake and fatty acid oxidation with resulting improvement of glucose homeostasis and, due to a reduction in intra-myocyte triglyceride accumulation, to improved insulin action. Finally, the increase in energy expenditure should lead to a decrease in body weight. The combination of these effects in the metabolic syndrome would be expected to significantly reduce the risk of developing cardiovascular diseases. Several studies in rodents support this hypothesis (Bergeron R. et al. “Effect of 5-aminoimidazole-4-carboxamide-1(beta)-D-rifuranoside infusion on in vivo glucose metabolism in lean and obese Zucker rats”, Diabetes, 50, 1076 (2001), Song S. M. et al. “5-aminoimidazole-4-dicarboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabeted (ob/ob) mice”, Diabetologia, 45, 56 (2002), Halseth A. E. et al. “Acute and chronic treatment of ob/ob and db/db mice with AICAR decreases blood glucose concentrations”, Biochem. and Biophys. Res. Comm., 294, 798 (2002), Buhl E. S. et al. “Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying feature of the insulin resistance syndrome”, Diabetes, 51, 2199 (2002)). Until recently, most in vivo studies relied on AICAR AMPK activator, a cell permeable precursor of ZMP. ZMP, a structural analogue of AMP, acts as an intracellular AMP mimic and, when accumulated to high enough levels, is able to stimulate AMPK activity (Corton J. M. et al. “5-aminoimidazole-4-dicarboxamide ribonucleoside, a specific method for activating AMP-activated protein kinase in intact cells?”, Eur. J. Biochem., 229, 558 (1995)). However, ZMP also acts as an AMP mimic in the regulation of other enzymes, and is therefore not a specific AMPK activator (Musi N. and Goodyear L. J., “Targeting the AMP-activated protein kinase for the treatment of type 2 diabetes”, Current Drug Targets-immune, Endocrine and Metabolic Disorders, 2 119 (2002)). Several in vivo studies have demonstrated beneficial effects of both acute and chronic AICAR administrations in rodent models of obesity and type 2 diabetes (Bergeron R. et al. “Effect of 5-aminoimidazole-4-carboximide-1b-D ribofuranoside infusion on in vivo glucose metabolism in lean and obese Zucker rats”, Diabetes, 50, 1076, (2001), Song S. M. et al. “5-aminoimidazole-4-carboxamide ribonucleotide treatment improves glucose homeostasis in insulin resistant diabetic (ob/bo) mice”, Diabetologia, 45, 56, (2002), Halseth A. E. et al. “Acute and chronic treatment of ob/ob and db/db mice with AICAR decreases blood glucose concentrations” Biochem. Biophys. Res. Comm. 294, 798, (2002), Buhl E. S. et al. “Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying feature of the insulin resistance syndrome”, Diabetes, 51, 2199 (2002)). For example, 7 week AICAR administration in the obese Zucker (fa/fa) rat leads to a reduction in plasma triglycerides and free fatty acids, an increase in HDL cholesterol, and a normalisation of glucose metabolism as assessed by an oral glucose tolerance test (Minokoshi Y. et al. “Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase”, Nature, 415, 339, -2002)). In both ob/ob and db/db mice, 8 day AICAR administration reduces blood glucose by 35% (Halseth A. E. et al. “Acute and chronic treatment of ob/ob and db/db mice with AICAR decreases blood glucose concentrations”, Biochem. Biophys. Res. Comm., 294, 798 (2002)). In addition to AICAR, it was found that the diabetes drug metformin can activate AMPK in vivo at high concentrations (Zhou G. et al. “Role of AMP-activated protein kinase in mechanism of metformin action”, J. Clin. Invest., 108, 1167, (2001), Musi N. et al. “Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes”, Diabetes, 51, 2074, (2002)), although it has to be determined to what extent its antidiabetic action relies on this activation. As with leptin and adiponectin, the stimulatory effect of metformin is indirect via activation of an upstream kinase (Zhou G. et al. “Role of AMP-activated protein kinase in mechanism of metformin action”, J. Clin. Invest., 108, 1167, (2001)). More recently, a small molecule AMPK activator has been described. This direct AMPK activator, named A-769662, is a thienopyridone and induces in vivo a decrease in plasma levels of glucose and triglycerides (Cool B. et al. “Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome”, Cell Metab., 3, 403-416, (2006)).
In addition to pharmacological intervention, several transgenic mice models have been developed in the last years, and initial results are currently becoming available. Expression of dominant negative AMPK in skeletal muscle of transgenic mice demonstrated the effect of AICAR on stimulation of glucose transport is dependent on AMPK activation (Mu J. et al. “Role for AMP-activated protein kinase in contraction and hypoxia regulated glucose transport in skeletal muscle”, Molecular Cell, 7, 1085, (2001)), and therefore likely not caused by non-specific ZMP effects. Similar studies in other tissues will help to further define the consequences of AMPK activation. It is expected that pharmacological activation of AMPK will have benefits in the metabolic syndrome with improved glucose and lipid metabolisms and reduction in body weight. In order to qualify a patient as having metabolic syndrome, three out of the five following criteria must be met:                1) elevated blood pressure (above 130/85 mmHg),        2) fasting blood glucose above 110 mg/dl,        3) abdominal obesity above 40″ (men) or 35″ (women) waist circumference, and blood lipid changes as defined by        4) increase in triglycerides above 150 mg/dl, or        5) decrease in HDL cholesterol below 40 mg/dl (men) or 50 mg/dl (women).        
Therefore, the combined effects that may be achieved through activation of AMPK in a patient who is qualified as having metabolic syndrome would raise the interest of this target.
Stimulation of AMPK has been shown to stimulate expression of uncoupling protein 3 (UCP3) skeletal muscle (Zhou M. et al. “UCP-3 expression in skeletal muscle: effects of exercise, hypoxia, and AMP-activated protein kinase”, Am. J. Physiol. Endocrinol. Metab., 279, E622, (2000)) and might therefore be a way to prevent from damage from reactive oxygen species. Endothelial NO synthase (eNOS) has been shown to be activated through AMPK mediated phosphorylation (Chen Z.-P. et al. “AMP-activated protein kinase phosphorylation of endothelial NO synthase”, FEBS Letters, 443, 285, (1999)), therefore AMPK activation can be used to improve local circulatory systems.
AMPK has a role in regulating the mTOR pathway. mTOR is a serine/threonine kinase and is a key regulator of protein synthesis. To inhibit cell growth and protect cells from apoptosis induced by glucose starvation, AMPK phosphorylates TSC2 at Thr-1227 and Ser-1345, increasing the activity of the TSC1 and TSC-2 complexes to inhibit m-TOR. In addition, AMPK inhibits mTOR action by phosphorylation on Thr-2446. Thus, AMPK indirectly and directly inhibits the activity of mTOR to limit protein synthesis. AMPK may also be a therapeutic target for many cancers that have constitutive activation of the PI3K-Akt signalling pathway. Treatment of various cancer cell lines by AICAR attenuated the cell proliferation both in in vitro and in vivo studies (Giri R., “5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase (AMPK)”, J. Biol. Chem. (2005)). Two reports link the treatment with metformin with a lower risk of cancer in diabetic patients (Evans J. M. “Metformin and reduced risk of cancer in diabetic patients”, BMJ, 330, 1304-1305, (2005)).
Activation of AMPK by AICAR has been shown to reduce expression of the lipogenic enzymes FAS and ACC, resulting in suppression of proliferation in prostate cancer cells. Many cancer cells display a markedly increased rate of de novo fatty acid synthesis correlated with high levels of FAS. Inhibition of FAS suppresses cancer cell proliferation and induces cell death. Thus, AMPK activation and inhibition of FAS activity is a clear target for pharmacological therapy of cancers.
In some publications it has been described that AICAR as an AMPK activator exerts anti-inflammatory diseases. It has been observed that AICAR attenuates the production of proinflammatory cytokines and mediators (S. Giri et al. J. Neuroscience 2004, 24:479-487), AICAR in rat model and in vitro attenuates EAE progression by limiting infiltration of leucocytes across blood brain barrier (BBB) (Nath. N. et al. J. of Immunology 2005, 175:566-574; Prasad R. et al. J. Neurosci Res. 2006, 84:614-625) and it has been suggested recently that AMPK activating agents act as anti-inflammatory agents and can hold a therapeutic potential in Krabbe disease/twitcher disease (an inherited neurological disorder) (S. Giri et al. J. Neurochem. 2008, Mar. 19).